The complete characterization of antibody-based drugs at the various stages of research and development still represents a major challenge for the biopharmaceutical industry especially with the emergence of next-generation biotherapeutic formats (Fc-fusion proteins, multispecific hybrids, …) with high molecular heterogeneity. One approach to achieve a detailed characterization is based on the analysis of biotherapeutics subunits using so-called middle-down mass spectrometry. These subunits are obtained by specific enzymatic digestions in the hinge region (IdeS, IgdE, BdpK, …) and/or by reduction and usually analyzed by classical reverse-phase chromatography coupled to mass spectrometry (RPLC-MS). Monoclonal antibody (mAb) subunits are in most cases well separated by RPLC, but this is not necessarily the case for the heterogeneous subunits of new biotherapeutics. Several mitigation strategies could be proposed such as longer gradients, 2D orthogonal LC separations (ion-exchange, size-exclusion, …), or even the non-chromatographic SampleStreamTM platform. However, each of these strategies presents drawbacks in terms of throughput, LC instrumentation and/or costs. The work described here aims at combining an online buffer exchange (OBE) performed on a classical Vanquish LC system with a high field asymmetric waveform ion mobility separation (FAIMS) on an Orbitrap Exploris 480 to achieve a higher analytical throughput. The OBE allows a rapid desalting of the intact construct or its subunits and the FAIMS leads to the separation of the co-eluting subunits prior to their analysis in the mass spectrometer.

Experiments were performed on several mAb and multispecific hybrid subunits in both denaturing and native ionization conditions to evaluate the resolving power of the FAIMS with different charge envelopes/conformations. This OBE-FAIMS-MS system intends to increase the analytical throughput and thus to propose an alternative to classical RPLC and native SEC analysis.